This invention relates to a vaccine composition for intranasal administration comprising one or more influenza antigens, and a chitosan as a mucosal adjuvant. The invention also relates to methods of immunization using the vaccine compositions, and to the use of certain chitosans for enhancing the immunogenicity of influenza viral antigens, when administered intranasally.
Current influenza vaccines consist of either inactivated whole virus, disrupted virus (split vaccines) or purified preparations of the membrane glycoproteins haemagglutinin (HA) and neuraminidase (NA) sub-unit vaccines. Haemagglutinin and neuraminidase are the antigens to which protective antibody responses are directed, haemagglutinin being the major protective antigen. Estimates of the efficacy of these parenterally administered vaccines vary greatly. Such vaccines are believed to act primarily by eliciting circulating anti-haemagglutinin IgG antibodies that transudate into the lower respiratory tract.
M. L. Clements et al, J. Clinical Microbiology 24, 157-160, 1986, have previously reported that both secretory IgA and serum IgG participate in immunity to influenza virus. Moreover, in mice, a number of published studies have demonstrated the importance of respiratory IgA to protection against influenza infection. It has also been found that an advantage of stimulating a local IgA response to influenza is that it is often of a broader specificity than the serum response and thus can provide cross-protection against viruses possessing haemagglutinin molecules different from those present in the vaccine. Accordingly, influenza vaccines that elicit both local secretory and serum anti-haemagglutinin responses should provide superior immunity to current vaccines. However, parenteral vaccination (intramuscular, sub-cutaneous etc.) is not effective at eliciting local antibody production, if there has been no previous mucosal exposure (e.g. infection). In order to stimulate the mucosal immune system, the vaccine must be applied topically to a mucosal surface.
Mucosal administration of influenza vaccine would have a number of advantages over traditional parenteral immunization regimes. Paramount amongst these are more effective stimulation of the local mucosal immune system of the respiratory tract and the likelihood that vaccine uptake rates would be increased because the fear and discomfort associated with injections would be avoided. Accordingly, a number of attempts have been made to develop mucosal influenza vaccines. A drawback however is that inactivated vaccines are often poorly immunogenic when given mucosally. For example, Kuno-sakai et al (vaccine 12: 1303-1310, (1994) have shown that administration of inactivated vaccine to humans gave strong mucosal and serum antibody responses and was effective in preventing infection by live vaccine virus. However, in order to achieve such results, Kuno-sakai et al administered three times the commercially available dose, an approach which is not considered to be commercially viable. In order to overcome this problem, different approaches to improving the immunogenicity of flu vaccines given orally or intranasally have included the use of the B sub-unit of cholera toxin (CTB) as an adjuvant (see S. Tamura et al, vaccine, 6, 409, (1988), encapsulation of the vaccine in a variety of microspheres (see Z. Moldoveanu et al, J. Inf. Dis. 167, 85-90 (1993), and the use of live attenuated strains (see H. F. Maassab et al vaccines, Plotkin S. A. and Mortimer F. A. Jr (eds) W. B. Saunders Philadelphia p435 (1993). To date however, the aforementioned approaches to the problem of poor mucosal immunogenicity have not resulted in the development of any commercially practicable means of enhancing the immunogenicity of mucosally administered flu vaccines, so far as the present applicants are aware.
The aforementioned problems have been solved to a very considerable extent by the compositions described in our earlier patent applications numbers WO-A-96 10421 and PCT/GB96/02680 (WO-A-97 16208) which disclose the use of chitosans as mucosal adjuvants. However, despite the efficacy of the compositions disclosed in our earlier applications, there remains room for improvement, particularly with regard to the pharmaceutical properties of the compositions, such as their long term stability.
Chitosan is a linear polysaccharide formed from repeating beta (1-4 linked) N-acetyl-D-glucosamine and D-glucosamine units, and is derived from the partial deacetylation of chitin obtained from the shells of crustaceans. Chitosan is usually made commercially by a heterogeneous alkaline hydrolysis of chitin to give a product which possesses a random distribution of remaining acetyl moieties. The properties of chitosans depend upon inter alia the degree of deacetylation, and the molecular weight. Most commercially available chitosans contain a population of chitosan molecules of varying molecular weights and varying concentrations of the component N-acetyl-D-glucosamine and D-glucosamine groups. The immunological properties of chitosans are known to be linked to the ratio between the N-acetyl-D-glucosamine and D-glucosamine groups.
The use of chitosans in an immunological context has been disclosed in articles by J. Iida et al. Vaccine, Vol. 5, pp 270-273 and K. Nishimura et al. Vaccine, 1984, Vol. 2, 99 94-100. Iida et al. and Nishimura et al. both disclosed the results of tests on chitosans having 30% and 70% deacetylation.
EP-A-0 183556 discloses the use of chitin and chitosan oligomers containing from two to seven glycosidic units as immune potentiating agents. The oligomers disclosed in this document are either fully deacetylated or contain the full complement of N-acetyl groups. There are no disclosures of partially deacetylated compounds. The highest molecular weight chitosan oligomer disclosed in this document would have a molecular weight of approximately 1439, which is a tiny fraction of the molecular weights (typically 500,000) of commercially available chitosan polymers.
WO-A-90 09780 discloses the use of various polycationic substances, such as chitosans, as mucosal absorption enhancers. This document contains a specific example illustrating the use of the xe2x80x9cSea Cure +xe2x80x9d grade of chitosan (obtainable from Protan Biopolymer A/S, Drammen, Norway) as an intranasal absorption enhancer for insulin. However, there is no disclosure or suggestion in this document that chitosans may act as vaccine adjuvants. The polycationic substances disclosed in this document are essentially high molecular weight polymers and, although it is stated that the polymers can have molecular weights of as low as 10,000, it is preferred that the molecular weights are at least 100,000 or 200,000 and most preferably about 500,000.
In WO-A-97/20576, it is disclosed that chitosans can be used as mucosal adjuvants for a wide variety of antigens. It is also disclosed in WO-A-97/20576 that the chitosan can have a molecular weight between 10 kD and 500 kD, molecular weights of between 50 kD and 300 kD being preferred and molecular weights of 10 kD to 300 kD being more preferred. Although influenza compositions identical to those in our earlier application WO-A-96 10421 are exemplified, it is stated to be preferred that the antigen is not an influenza antigen, and there is no disclosure of influenza antigen compositions containing chitosans of less than 100,000 kD weight average molecular weight, nor is there any discussion as to the importance of molecular weight to solution stability.
In our earlier patent applications WO-A-96 10421 and PCT/GB96/02680 (WO-A-97 16208A), it is disclosed that by administering the haemagglutinin and neuraminidase antigens of influenza together with a particular chitosan derivative in an intranasal formulation, it is possible to achieve good IgG and good IgA responses. It has now been found that by selecting certain grades of chitosans, and in particular chitosans of defined molecular weights, it is possible to provide formulations having improved long term stability.
The efficacy of chitosans as adjuvants depends to a considerable extent on the extent of the level of deacetylation. Chitosans having only low levels of deacetylation have very poor adjuvant properties, at least insofar as influenza vaccine antigens are concerned. It has been found by the present inventors that for good adjuvant properties, the chitosan should be at least 80% deacetylated.
Chitosans having this level of deacetylation are relatively insoluble in water in the free base form and therefore are usually presented in the form of acid addition salts such as glutamate salts. Such salts can be formed by dissolving the free base form of the chitosan in acid and then drying. Because of the high proportion of free amino groups (which have a pKa of about 6.6) available for reaction with acids, acid addition salts of highly deacetylated chitosans are ad: significantly more acidic than chitosans having a lower level of deacetylation. Thus, acid addition salts of chitosans having approximately 85% deacetylation form solutions having a pH of about 4 to 5. However, a problem with preparing solutions at pH 4, particularly vaccine solutions containing influenza vaccine antigens, is that influenza antigens are susceptible to degradation and loss of activity at low pH, such as pH 4. Indeed, even at pH values as high as pH 6, it has been found by the applicants that the influenza antigens are relatively unstable on storage and that there is an appreciable loss of activity. It will be appreciated therefore that there is a conflict between the need, on the one hand, to optimise the adjuvant activity of the chitosan by using a highly deacetylated form, and the equally important need on the other hand to ensure stability of the antigen.
It is of course possible in principle to adjust the acidity of the vaccine solution to a pH nearer to neutral at which the antigens are more stable, but a further problem is that chitosan salts such as chitosan hydroglutamate tend to be only poorly soluble, if at all, at pH 7, and this creates problems with the stability of the formulation. In particular, during long term storage, there is a tendency for precipitation of the chitosan from solution to occur. Such precipitation is clearly unacceptable in a pharmaceutical context, particularly where vaccines are concerned.
The foregoing summary, as well as the following detailed description of embodiments of the invention, will be better understood when shown in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which may be preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.